Over the last two years, we continued to work on structural aspects of HIV envelope glycoproteins, which are molecular machines that mediate viral entry. In our first application of advanced methods for single particle processing, we carried out cryo-electron microscopic analysis at sub-nanometer resolution of a cleaved, soluble version of trimeric Env to explore structural changes that take place with ligand activation. We showed that Env in the open, activated conformation has highly conserved elements of gp41 organized in a three-helix motif in the central portion of the envelope glycoprotein complex. The N-terminal gp41 helices in this novel, activated Env conformation are much less compactly packed than in the post-fusion, six-helix bundle state, suggesting a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage. Extending this work to the closed, pre-fusion state of trimeric Env led to an important discovery. A cryo-electron microscopic structure of the closed, pre-fusion state of trimeric HIV-1 Env in complex with the broadly neutralizing antibody VRC03, determined to a resolution of 6 Angstrom, showed that three gp41 helices at the core of the trimer serve as an anchor around which the rest of Env is reorganized upon activation and transition to the open quaternary conformation. The overall organization of trimeric HIV-1 Env, which we have now determined in both pre-fusion and activated intermediate states, bears a striking resemblance to the structures previously observed for influenza hemagglutinin trimers. The possible similarity in spike architecture and activation mechanism between HIV, influenza and other viruses such as Ebola has long been hypothesized. In influenza hemagglutinin, three copies of HA1 are arranged around the central stalk formed by HA2 trimers in the pre-fusion state. Movement of HA1 protomers away from the central axis during the activation process ultimately leads to formation of the post-fusion six-bundle state formed from HA2 trimers. The role of co-receptor binding in the case of the HIV-1 spike is most likely analogous to the role of low pH in induction of conformational changes in the influenza spike, but how these changes lead to the formation of the pre-hairpin intermediate state, and ultimately to membrane fusion, is still not understood in mechanistic detail. Our findings with HIV-1 Env showing a closed pre-fusion state with a central stalk made up of three alpha-helices and an activation process that leads to an outward displacement of the three gp120 protomers thus provides further and strong evidence for a fundamental similarity between HIV and influenza, extending previously noted similarities in the metastability of the pre-fusion state and the architecture of the final post fusion state. Considering all of these structural and functional similarities in the fusion machinery, it is not surprising that the broadest human monoclonal antibodies against both HIV and influenza target gp41 and HA2. Application of emerging methods in single particle cryo-electron microscopy (cryo-EM) to large complexes, mostly with high symmetry, to has led to the determination of the structures of a variety of macromolecular complexes at sub-nanometer resolutions. Extending cryo-EM methods to routinely determine the structures of small macromolecular complexes with sizes of 500 kDa at sub-nanometer resolution still remains an exciting frontier in modern structural biology. As part of CRADA collaboration with FEI Company, we have made important advances to establish step-wise systematic workflows for structure determination by cryo-EM at sub-nanometer resolution. Inspection of the distribution of the released entries belonging to the single particle category in the EM data bank at the end of 2012 revealed that the vast majority of structures at 10 Angstrom or better resolution are from large protein complexes, with only six examples (representing four distinct protein complexes) below 500 kDa, reflecting smaller, potentially dynamic protein complexes. The great growth in this field over the last year is reflected by the fact that the number of entries in this category has more than doubled in just a year. These types of complexes are at the heart of cell function, and understanding how they work will generate fundamental insights that will be key to the development of biomedical therapeutics in the coming decades. It is safe to predict that addressing this critical gap in the landscape of structural biology is where we may expect the greatest future impact of cryo-EM. The use of automated cryo-EM techniques could prove to be useful in general for rapid determination of drug binding sites on proteins and protein complexes, especially in cases where only small amount of proteins are available, or when there are levels of aggregation that are incompatible with the use of X-ray crystallography or when answers are needed more quickly than the time that it takes to determine structures using X-ray crystallography.